Construction and characterization of a bacterial artificial chromosome library of peach

Abstract 

A peach [Prunus persica (L.) Batch] bacterial artificial chromosome (BAC) library of var. Jingyu was constructed. Jingyu is a traditional variety, that displays many of the important agronomic characters of stone fruits. Since peach leaves are rich in polysaccharides, high-molecular-weight (HMW) DNA was extracted from leaf nuclei using a protocol adapted to peach. The HMW DNA embedded in agarose plugs was partially digested by HindIII. After size-selection by pulsed field gel electrophoresis, the selected DNA fragments were ligated to pBeloBAC11 and transformed into E. coli DH10B cells by electroporation. In total 20,736 recombinant clones were obtained. The BAC library has an average insert size of 95 kb and represents approximately 6.7 peach haploid genome equivalents. The BAC clones were stable in E. coli cell after 100 generations. The lack of hybridization to chloroplast and mitochondrial genes demonstrated that the library is predominantly composed of nuclear DNA. The library was screened with two molecular markers, W4 and P20, that are linked to white flesh and nectarine genes of peach, respectively. Ten positive clones were detected. Their fingerprints will be used to determine clone relationships and assemble contigs. This library should be well-suited for the map-based cloning of peach genes and genome physical mapping.